Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

Morbidity and Mortality Associated with Gastrectomy for Gastric Cancer

Background

Surgery alone is often inadequate for advanced-stage gastric cancer. Surgical complications may delay adjuvant therapy. Understanding these complications is needed for multidisciplinary planning.

Material and Methods

The American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database was queried for patients who underwent gastrectomy for malignancy (ICD-9 code 151.x) from 2005 to 2010. Thirty-day mortality and morbidity were evaluated.

Results

Overall, 2,580 patients underwent gastrectomy for malignancy, divided as total gastrectomy 999 (38.7 %) and partial gastrectomy 1,581 (61.3 %). Overall, serious morbidity occurred in 23.6 %, and the 30-day mortality was 4.1 %. Patients receiving a total gastrectomy were younger and healthier than those receiving a partial gastrectomy for the following measured criteria: age, diabetes, chronic obstructive pulmonary disease and hypertension. Serious morbidity and mortality were significantly higher in the total gastrectomy group than the partial gastrectomy group (29.3 vs. 19.9 %, p < 0.001; and 5.4 vs. 3.4 %, p < 0.015, respectively). The inclusion of additional procedures increased the risk of mortality for the following: splenectomy (odds ratio [OR] 2.8; p < 0.001), pancreatectomy (OR 3.5; p = 0.001), colectomy (OR 3.6; p < 0.001), enterectomy (OR 2.7; p = 0.030), esophagectomy (OR 3.5; p = 0.035). Abdominal lymphadenectomy was not associated with increased morbidity (OR 1.1; p = 0.41); rather, it was associated with decreased mortality (OR 0.468; p = 0.028).

Conclusions

Gastrectomy for cancer as currently practiced carries significant morbidity and mortality. Inclusion of additional major procedures increases these risks. The addition of lymphadenectomy was not associated with increased morbidity or mortality. Strategies are needed to optimize surgical outcomes to ensure delivery of multimodality therapy for advanced-stage disease.     

Perturbations in the erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine

In vivo observations on the kinetics of F cells and of fetal hemoglobin (HbF) synthesis and in vitro studies of erythroid progenitors, their number, and the gamma-gene expression in their progeny were carried out in baboons (Papio cynocephalus) treated with 5-azacytidine. Maximum effect on the increase of HbF production in vivo was observed only when an expanded erythroid marrow population was present. In these animals, as well as in normal animals, treatment resulted in a significant reduction of the late erythroid progenitor cell pools (erythroid clusters and erythroid colony-forming units, CFU-E) in the marrow. This reduction was more pronounced among those progenitors grown in the absence of added erythropoietin, and it was followed by a rebound a few days after treatment cessation, reflecting the accumulation of regenerating progenitors. An early increase in the in vitro synthesis of HbF in erythroid clusters and CFU-E colonies was observed. This increase was further documented at the cellular level, with immunofluorescent labeling of colonies with monoclonal anti-gamma- globin chain antibodies. In contrast to the findings in late progenitors, the number of erythroid burst-forming unit (BFU-E) colonies and the synthesis of HbF in these colonies was not influenced significantly by 5-azacytidine treatment. It is proposed that the toxic effects of 5-azacytidine on late progenitors, leading to faster mobilization of earlier progenitors to the next more mature compartment, play a role in the in vivo augmentation of HbF synthesis by this drug. This perturbation in the progenitor cell population kinetics and the presumed hypomethylation of the surviving differentiating cells may act synergistically to produce a maximum HbF response after 5-azacytidine treatment.     

Human Calcitonin Delivery in Rats by Iontophoresis

Abstract— In-vitro iontophoresis (0·33 mA cm⁻²) of calcitonin (50 μg mL⁻¹, pH 4) was performed with the hairless rat skin model. Direct current was as potent as pulse current (2·5 kHz on/off 1/1) iontophoresis in promoting transdermal permeation of calcitonin. Increase in duration of current application from 20 min to 1 h did not increase calcitonin flux. Results suggest that calcitonin can be blocked in the skin pores through which it travels or can accumulate in the skin and be progressively released from the depot. Invivo experiments showed that transdermal iontophoretic administration of calcitonin induced a hypocalcaemic effect in rats.     

The efficacy of postoperative pain management with ketorolac for day case oral surgery

The efficacy of the non-steroidal anti-inflammatory analgesic, ketorolac (Toradol), was investigated in 52 day case patients undergoing removal of impacted third molar teeth under intravenous sedation and local analgesia. The study was double-blind, randomized and placebo-controlled. A single 30 mg dose of ketorolac was administered intravenously just prior to induction of sedation with midazolam. Ketorolac was well tolerated and provided good postoperative analgesia. It is suggested that ketorolac is a useful addition to the analgesic armamentarium and appropriately prescribed, provides good pain relief following day case oral surgery.     

Urinary C-peptide creatinine ratio to detect absolute insulin deficiency in type 2 diabetes

BackgroundNational Institute for Health and Clinical Excellence guidelines (CG87) recommend neutral protamine hagedorn (NPH) insulin for the provision of basal insulin in type 2 diabetes, but use of analogue insulin is as much as 40%. Where residual endogenous insulin secretory capacity is present there is no evidence that analogue insulins provide any additional benefit over human insulins, and they come at an expensive premium. Anecdotally, however, there is a reluctance to switch people back to NPH insulin, partly because of a perceived risk of pancreatic failure and potential ketosis. Urinary C-peptide creatinine ratio (UCPCR) has been validated as a method for evaluating residual endogenous insulin secretion in type 1 and type 2 diabetes, with a UCPCR of no more than 0·2 nmol/mmol suggestive of absolute insulin deficiency. We aimed to evaluate the prevalence of true insulin deficiency among patients with type 2 diabetes with UCPCR, and confirm findings with the gold standard mixed meal tolerance test (MMTT).Methods191 insulin-treated patients with a clinical diagnosis of type 2 diabetes (diagnosed at or after age 45 years and who did not start insulin within the first year of diagnosis) collected a 2-h post-prandial urine sample for UCPCR measurement. Nine patients from two subgroups (UCPCR ≤0·2 nmol/mmol and UCPCR >0·2) completed a standard MMTT.Findings11 (5·8%) of 191 patients had two consistent UCPCRs of less than or equal to 0·2 nmol/mmol. Nine were able to do the MMTT, of whom five were confirmed to have absolute insulin deficiency (stimulated serum c-peptide <0·2 nmol/L). Three of these five patients were glutamic acid decarboxylase antibody-negative. Nine of nine patients with UCPCR of more than 0·2 nmol/L had confirmed endogenous insulin secretion in their MMTT. Those with insulin deficiency had a shorter time to starting insulin (median 2·5 years [IQR 1·5–3·0] vs 6·0 [3·0–10·75], p=0·005) and lower body-mass index (25 kg/m² vs 29, p=0·04) but no other significant differences in clinical characteristics.InterpretationWe have demonstrated a very low prevalence of true pancreatic failure in this population of insulin-treated patients with type 2 diabetes. This requires further exploration by comparison of a population being treated with NPH insulin with one on analogue insulin, and then determining whether UCPCR could act as a clinical decision support tool to safely switch from analogue insulin to NPH insulin.FundingNational Institute for Health Research.     

Human papillomavirus prevalence and type distribution in male anogenital sites and semen.

BACKGROUND: Human papillomavirus (HPV) is sexually transmitted and causes cervical cancer. Although HPV can infect men and women, little is known about infection in men. Specifically, the prevalence of type-specific HPV infection and the distribution of infections by anogenital anatomic site in men are incompletely characterized. METHODS: We tested 463 men ages 18 to 40 years for HPV at the glans/corona, penile shaft, scrotum, urethra, perianal area, anal canal, and in a semen sample. Eligible men acknowledged no history of genital warts and had sexual intercourse with a woman within the past year. HPV testing by PCR and reverse line blot genotyping for 37 types was conducted on each of the specimens from the seven sampling sites. RESULTS: When HPV results from any sampling site were considered, 237 (51.2%) men were positive for at least one oncogenic or nononcogenic HPV type, and another 66 (14.3%) men were positive for an unclassified HPV type. The types with the highest prevalence were HPV-16 (11.4%) and 84 (10.6%). External genital samples (glans/corona, shaft, and scrotum) were more likely than anal samples to contain oncogenic HPV (25.1% versus 5.0%). HPV-positive penile shaft and glans/corona samples were also more likely to be infected with multiple HPV types than other sites. CONCLUSIONS: More complete anogenital sampling and sensitive detection for 37 HPV types resulted in a higher HPV prevalence in primarily asymptomatic men than reported previously. The penile shaft was the site most likely to be HPV positive and harbored the greatest proportion of multiple type and oncogenic infections. These results have implications for research of HPV among men and transmission between partners.     

Randomized controlled trial of dexamethasone treatment in very-low-birth-weight infants with ventilator-dependent chronic lung disease

This randomized controlled trial was designed to answer the question: does administration of dexamethasone to neonates with bronchopulmonary dysplasia decrease the need for assisted ventilation? Twenty-five infants with a birth weight < 1501 g, requiring mechanical ventilation and FiO₂ of ± 0.30 at 21-35 days of age, were randomized to treatment with iv dexamethasone or to sham injections for 12 days. The primary outcome criterion was extubation within seven days after study entry. Treatment (n= 12) and control (n= 13) groups were well matched at entry. Dexamethasone facilitated weaning from assisted ventilation (p= 0.0154). There was no increased incidence of infection. Dexamethasone treatment resulted in a significant increase in glucosuria (p= 0.0002) and in systolic blood pressure (p= 0.0034). There was a significant decrease in heart rate (p= 0.0001) and a significant weight loss (p= 0.0002) following dexamethasone treatment. Dexamethasone treatment facilitated weaning from assisted ventilation but several systemic effects were noted that deserve further evaluation before dexamethasone becomes routine treatment.